The single-stranded DNA is a crucial and active intermediate in the processes of DNA replication, recombination, and repair, which are essential for the transmission of genetic information. This intermediate is formed by a transient unwinding of the duplex DNA, which is catalyzed by a class of enzymes called helicases. The helicases are indispensable in all aspects of DNA and RNA metabolism, serving also as molecular pumps and as biological motors for large multiple-protein machines, including the replisome and pre-primosome. Genomic DNA damages, due to environmental and cellular factors, are constantly occurring in the cell, leading to the formation of the stalled replication fork. The restart of the stalled fork occurs through the assembly of the large molecular machine, the pre-primosome, which is a key process in defending the integrity of the genetic information. In E. coli, two helicases, the DnaB and PriA proteins, together with the DnaC, DnaT, PriB, and PriC proteins, are engaged in the pre-primosome assembly and functions. Elucidation of the helicase mechanisms is of paramount importance for understanding the fundamental processes of the nucleic acid metabolism and why such processes dysfunction in, e.g., cancer and human genetic diseases. Studying the molecular mechanisms will provide the necessary knowledge as to how to regulate and control these processes and is invaluable in designing efficient therapies for diseases. Elucidation of the mechanisms of the pre-primosome assembly and activities is crucial for understanding the rules governing the restart of the stalled fork and, in general, the functioning of large molecular machines. The E. coli DnaB protein is a paradigm model of a hexameric replicative helicase. Hexameric replicative helicases were originally classified as the DnaB-like family of enzymes. The primosome is an archetype molecular system for the collaborative action of different motor proteins and of a large molecular machine. The PriA protein is, in turn, a prototype model for the factor, which senses the presence of the stalled fork and initiates the assembly of the pre-primosome. The long-term goal of this project is to establish molecular mechanisms of replicative helicases and their engagement in the pre-primosome, and the rules, which govern the macromolecular interactions in the pre- primosome machine. This will be accomplished through quantitative studies of the thermodynamics, kinetics, and structures of multiple protein-protein and protein-DNA complexes, using the analytical ultracentrifugation, dynamic light scattering, chemical rapid quench-flow, fluorescence stopped-flow, fluorescence anisotropy, fluorescence energy transfer, crystallography, electron microscopy, and biochemical methods.